microPublication Biology 2578-9430 Caltech Library 10.17912/micropub.biology.002229 new finding gene model drosophila Gene model for the ortholog of sad in Drosophila cardini Patel Payal Data curation Formal analysis Investigation Writing - review & editing 1 Chialvo Pablo Investigation Formal analysis Writing - review & editing Validation 1 Scott Chialvo Clare Conceptualization Supervision Validation Writing - original draft 1 § Biology, App State, Boone, NC, US Correspondence to: Clare Scott Chialvo ( chialvoch@appstate.edu )

The authors declare that there are no conflicts of interest present.

 25 6 2026 2026 2026 10.17912/micropub.biology.002229 3 6 2026 24 6 2026 25 6 2026 Copyright: © 2026 by the authors 2026 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

We developed a gene model for the shadow ortholog ( sad ) in the ASM1890373v1 Genome Assembly (GenBank Accession: GCA_018903735.1) of Drosophila cardini . This ortholog was characterized as part of a developing dataset for a comparative study of detoxification gene family evolution in the immigrans - tripunctata radiation of the genus Drosophila using an adapted Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

This gene annotation project was funded by Nation Science Foundation grants DEB-1737869 (PI LKR, CoPI CSC) and DBI-2217912 (PI CSC). The Genomics Education Partnership (GEP; https://thegep.org/ ), which supports this project, is funded by the National Science Foundation (1915544; PI LKR) and the National Institute of General Medical Sciences of the National Institutes of Health (R25GM130517; PI LKR). Any opinions, findings, and conclusions or recommendations expressed in this material are solely those of the author(s) and do not necessarily reflect the official views of the National Science Foundation nor the National Institutes of Health.

(A) Synteny comparison of the genomic neighborhoods for sad in Drosophila melanogaster and D. cardini . Thin underlying arrows indicate which DNA strand the target gene, sad , is located on in D. melanogaster (top) and D. cardini (bottom). The thin arrow pointing to the right indicates that sad is on the positive strand in D. melanogaster , and the thin arrow pointing to the left indicates that sad is on the negative strand in D. cardini . The wide gene arrows pointing in the same direction as sad are on the same strand relative to the thin underlying arrows, while wide gene arrows pointing in the opposite direction of sad are on the opposite strand relative to the thin underlying arrows. White gene arrows in D. cardini indicate orthology to the corresponding gene in D. melanogaster , while black gene arrows indicate non-orthology. Gene symbols given in the D. cardini gene arrows indicate the orthologous gene in D. melanogaster , while the locus identifiers are specific to D. cardini . (B) Gene Model in GEP UCSC Track Data Hub (Raney et al., 2014). The coding-regions of sad in D. cardini are displayed in the User Supplied Track (red); coding sequences (CDS) are depicted by thick rectangles and introns by thin lines with arrows indicating the direction of transcription. Subsequent evidence tracks include Spaln of D. melanogaster Proteins (purple, alignment of Ref-Seq proteins from D. melanogaster ), Coding Regions Predicted by Augustus (blue), GeMoMa (teal), and NSCAN PASA-EST (dark green), and RNA-Seq from mixed sex adult flies (brown; alignment of Illumina RNA-Seq reads from D. cardini – Erlenbach et al. 2023). (C) Dot Plot of sad-PA in D. melanogaster ( x -axis) vs. the orthologous peptide in D. cardini ( y -axis). Amino acid number is indicated along the left and bottom; CDS number is indicated along the top and right, and CDSs are also highlighted with alternating colors. Line breaks in the dot plot indicate areas of with low amino acid sequence identity and/or gaps between species. In CDS 1, there is a long break (purple box – a), and in CDS 3 there is a shorter break (light blue box – b). (D) Idiosyncrasies in protein alignment. CDS 1 contains one long break in the protein alignment and CDS 3 contains a shorter break in the protein alignment that indicate low levels of sequence similarity. In the CDS 1 break (purple box – a), two gaps were added to the D. cardini sequence to align it with the D. melanogaster sequence, and there are areas where the sequence includes several amino acids that are chemically dissimilar. The CDS 3 break (light blue box – b), includes the addition of two gaps to the D. cardini sequence and several dissimilar amino acids.

Description

Introduction

This article reports a predicted gene model generated by undergraduate work using a structured gene model annotation protocol defined by the Genomics Education Partnership (GEP; thegep.org ) for Course-based Undergraduate Research Experience (CURE). The following information in this box may be repeated in other articles submitted by participants using the same GEP CURE protocol for annotating Drosophila species orthologs of Drosophila melanogaster detoxification genes.

“Within insects, the process of detoxifying xenobiotics and host secondary metabolites is a three-phase process that involves functionalization, conjugation, and excretion of these compounds. Expansions of known detoxification gene families ( e.g. , cytochrome P450s) is associated with diet breadth and insecticide resistance (Ranson et al., 2002; Després et al., 2007; Rane et al., 2016). With the increasing availability of high-quality genomes for non-model organisms, including Drosophila species beyond D. melanogaster , it is now possible to perform large scale comparative studies (Robinson et al., 2011; Kim et al., 2021; Threfall and Baxter, 2021). Careful manual annotation and curation of gene models can improve upon computational gene predictions in non-model species, which aids the accuracy of studies on gene and genome evolution (Mudge and Harrow, 2016; Tello-Ruiz et al., 2019). To aid in these annotations, the Genomics Education Partnership (thegep.org) developed a curriculum involving web-based tools that allow undergraduates to engage in authentic course-based research focused on manually annotating genes in non-model species (Rele et al., 2023). The orthologous gene models, including the one presented here, then provide a reliable basis for further evolutionary genomic analyses when made available to the scientific community. The gene ortholog described here in D. cardini for shadow ( sad ), a member of the cytochrome P450 monooxygenase gene family, was characterized as part of a developing dataset for a comparative study of detoxification gene families in the immigrans - tripunctata radiation of the genus Drosophila .” (Williams et al., 2026)

In the subgenus Drosophila , D. cardini Sturtevant 1916 is a member of the cardini subgroup in the cardini species group of the immigrans-tripunctata radiation (Heed and Krishnamurthy, 1959; Bächli, 2005). Species in the cardini subgroup are found in the mainland Neotropics, and the range of D. cardini extends from Florida to Brazil (Heed, 1962). Members of the cardini group primarily feed and develop on fruit and flowers (Markow and O'Grady, 2008). However, D. cardini is also reported to feed on mushrooms and can tolerate the cyclopeptide toxin α-amanitin (Stump et al., 2011).

Cytochrome P450 monooxygenases (CYPs) are a family of phase I detoxification enzymes that are found in almost all aerobic organisms and act by oxidizing compounds to make them more polar (Stegeman and Livingstone, 1998; Li et al., 2007). The enzymes in this family vary in both their substrate specificity and the range of metabolites that they produce (Rendic and Di Carlo, 1997; Scott, 1999). Furthermore, the substrate specificity of CYPs can be altered by a change in a single amino acid (Lindberg and Negishi, 1989). A member of the Halloween gene group, shadow ( sad ) is a mitochondrial CYP whose expression is restricted to the prothoracic gland cells of the ring gland (Gilbert, 2004). Halloween genes act in the biosynthetic pathway that produces 20-Hydroxyecdysone, a hormone that controls metamorphosis and development in arthropods (Gilbert and Warren, 2005; Rewitz et al., 2006). sad mutants exhibit phenotypes similar to disembodied mutants and die prior to completing embryogenesis displaying abnormal cuticular development (Warren et al., 2002; Gilbert ,2004).

We propose a gene model for the D. cardini ortholog of the D. melanogaster shadow ( sad ) gene. The genomic region of the ortholog corresponds to the Augustus gene prediction JAEIGM010000023.g145.t1 in the ASM1890373v1 Genome Assembly of D. cardini (GCA_018903735.1 – Kim et al., 2021). This model is based on RNA-Seq data from D. cardini (Erlenbach et al. 2023; https://doi.org/10.5061/dryad.hdr7sqvq2 ) and sad in D. melanogaster using FlyBase release FB2024_02 (GCA_000001215.4; Gramates et al., 2022; Jenkins et al., 2022; Larkin et al., 2021).

Synteny - The reference gene, sad, occurs on chromosome 3R in D. melanogaster and is flanked upstream by CG6959 and Kynurenine aminotransferase ( Kyat ) and downstream by CG6962 , salsa ( salsa ), and methuselah-like 5 ( mthl5 ) which is nested in salsa . The tblastn search of D. melanogaster sad-PA (query) against the D. cardini (GenBank Accession: GCA_018903735.1 Genome Assembly (database) placed the putative ortholog of sad within contig_901 (JAEIGM010000023) which corresponds to Augustus gene prediction JAEIGM010000023.g145.t1 (E-value: 0.0; percent identity: 69.11% as determined by blastp ). The putative ortholog is flanked upstream by Augustus gene prediction JAEIGM010000023.g146.t1 and JAEIGM010000023.g147.t1, which correspond to CF transmembrane conductance regulator ( Cftr ) and Coiled-coil domain containing protein 39 ( Ccdc39 ) in D. melanogaster (E-value: 0.0 and 0.0 identity: 79.64% and 61.61%, respectively, as determined by blastp ; Figure 1A ; Altschul et al., 1990). The putative ortholog of sad is flanked downstream by Augustus gene prediction JAEIGM010000023.g144.t1 and GeMoMa gene predictions FBtr0082516_R0, FBtr0301042_R0, and FBtr82515_R0 (nested in FBtr0301042_R0, which correspond to Cftr , CG6962 , salsa , and mthl5 in D. melanogaster (E-value: 0.0 for all; identity: 64.05%, 66.19%, 86.34%, and 75.86%, respectively, as determined by blastp ). The putative ortholog assignment for sad in D. cardini is supported by the following evidence: The tblastn results are of good quality, and all coding sequences (CDS) and isoforms found in D. melanogaster also appear to be present in D. cardini . The gene predictions surrounding the sad ortholog are not fully conserved. CG6962 and salsa with mthl5 nested in it are downstream of sad as expected, but a copy of Cftr occurs between them and sad . Both of the upstream gene predictions, Cftr and Ccdc39 , occur on chromosome 3R in D. melanogaster . However, Cftr is much further upstream and Ccdc39 is downstream in D. melanogaster . This suggests the potential for multiple chromosomal inversions in this region. Inversions are common within the genus Drosophila and play an important role in speciation (Powell, 1997; Bhutkar et al., 2008; Reis et al., 2018). We conclude that the Augustus gene prediction JAEIGM010000023.g145.t1 is an ortholog of sad in D. cardini ( Figure 1A ).

Protein Model - sad in D. cardini has 6 CDSs within the genome sequence. The only unique protein sequence (sad-PA) is translated from 1 mRNA isoform (sad-RA; Figure 1B ). Relative to the ortholog in D. melanogaster , the CDS number and protein isoform count are conserved . The sequence of sad-PA in D. cardini has 67.9% identity (81.0% similarity) with the protein-coding isoform sad-PA in D. melanogaster , as determined by blastp ( Figure 1C ). This level of divergence is not surprising given that D. cardini and D. melanogaster belong to two separate subgenera ( Drosophila and Sophophora respectively) that diverged approximately 45-60MYA (Russo et al., 1995; Tamura et al., 2004; Obbard et al., 2012). Coordinates of this curated gene model are archived in the CaltechDATA repository (see “Extended Data” section below).

Methods

The annotation methods used in this project are adapted from those described in Rele et al. (2023), which includes algorithms, database versions, and citations for the complete annotation process developed for the Pathways Project. The methods for the current project are detailed in brief below with notes on significant differences between this protocol and the one described in Rele et al. (2023). The students use the GEP instance of the UCSC Genome Browser v.435 ( https://gander.wustl.edu ; Kent et al., 2002; Raney et al., 2024) to examine the genomic neighborhood of their reference detoxification gene in the D. melanogaster genome assembly (Aug. 2014; BDGP Release 6 + ISO1 MT/dm6). Students obtain the protein sequence for the D. melanogaster target gene for a given isoform and use a tblastn search of the sequence against their target Drosophila species genome assembly ( D. cardini (GCA_018903735.1 – Kim et al., 2021)) on the NCBI BLAST server ( https://blast.ncbi.nlm.nih.gov/Blast.cgi , Altschul et al., 1990) to identify the putative ortholog location. Students compare the genomic neighborhood of the putative ortholog to that of the reference gene in D. melanogaster . This local synteny analysis includes a minimum of two upstream and downstream genes relative to the potential ortholog. As no RefSeq protein data is available for these species, comparisons are based on gene predictions that correlate with gene expression data in the putative ortholog neighborhood. Using the multiple alignment tracks feature in the Genome Browser, students examine other sets of genomic evidence, including Spaln alignment of D. melanogaster proteins, multiple gene prediction tracks (e.g., GeMoMa, Augustus, NSCAN PASA-EST), and mixed sex adult RNA-Seq expression data from the target species generated by Erlenbach et al. (2023; https://doi.org/10.5061/dryad.hdr7sqvq2 ). Information on the genomic structure information (e.g., CDSs, intron-exon number, number of isoforms) for the reference gene in D. melanogaster is retrieved using Gene Record Finder ( https://gander.wustl.edu/~wilson/dmelgenerecord/index.html ; Rele et al ., 2023). To determine approximate splice sites within the target gene, a tblastn search using the CDSs from the D. melanogaste r reference gene against the putative ortholog location (10kb up- and downstream of the target gene prediction). Coordinates of the CDS(s) are refined by examining aligned RNA-Seq data, identifying canonical splice site sequences, and ensuring the maintenance of an open reading frame. Students confirm the biological validity of their target gene model using the FlySeq Gene Model Checker ( https://gander2.wustl.edu/~wilson/genechecker-flyseq/ ), which compares the hypothesized target gene model's structure and translated sequence against the D. melanogaster reference gene. At least two independent models for this gene are generated. These models are reconciled by a third independent researcher to produce the final model presented here. Note: comparison of 5' and 3' UTR sequence information is not included in this GEP CURE protocol.

Extended Data

Description: Zipped archive containing FASTA, PEP, and GFF files for sad model. Resource Type: Model. DOI: https://doi.org/10.22002/0jmvb-n8f52

We would like to thank Wilson Leung for developing and maintaining the technological infrastructure that was used to create this gene model and Laura K. Reed for overseeing the Genomics Education Partnership. Thank you to FlyBase for providing the definitive database for Drosophila melanogaster gene models. FlyBase is supported by grants: NHGRI U41HG000739 and U24HG010859, UK Medical Research Council MR/W024233/1, NSF 2035515 and 2039324, BBSRC BB/T014008/1, and Wellcome Trust PLM13398.

Altschul SF Gish W Miller W Myers EW Lipman DJ 1990 10 5 Basic local alignment search tool. J Mol Biol 215 3 0022-2836 403 410 10.1016/S0022-2836(05)80360-2 2231712 Bächli, G. (2005) Taxodros: The database on taxonomy of Drosophilidae, version February 2026, last accessed 28 May 2026. https://taxodros.uzh.ch/ Bhutkar A Schaeffer SW Russo SM Xu M Smith TF Gelbart WM 2008 7 13 Chromosomal rearrangement inferred from comparisons of 12 Drosophila genomes. Genetics 179 3 0016-6731 1657 1680 10.1534/genetics.107.086108 18622036 Després L David JP Gallet C 2007 2 26 The evolutionary ecology of insect resistance to plant chemicals. Trends Ecol Evol 22 6 0169-5347 298 307 10.1016/j.tree.2007.02.010 17324485 Drosophila 12 Genomes Consortium. Clark AG Eisen MB Smith DR Bergman CM Oliver B Markow TA Kaufman TC Kellis M Gelbart W Iyer VN Pollard DA Sackton TB Larracuente AM Singh ND Abad JP Abt DN Adryan B Aguade M Akashi H Anderson WW Aquadro CF Ardell DH Arguello R Artieri CG Barbash DA Barker D Barsanti P Batterham P Batzoglou S Begun D Bhutkar A Blanco E Bosak SA Bradley RK Brand AD Brent MR Brooks AN Brown RH Butlin RK Caggese C Calvi BR Bernardo de Carvalho A Caspi A Castrezana S Celniker SE Chang JL Chapple C Chatterji S Chinwalla A Civetta A Clifton SW Comeron JM Costello JC Coyne JA Daub J David RG Delcher AL Delehaunty K Do CB Ebling H Edwards K Eickbush T Evans JD Filipski A Findeiss S Freyhult E Fulton L Fulton R Garcia AC Gardiner A Garfield DA Garvin BE Gibson G Gilbert D Gnerre S Godfrey J Good R Gotea V Gravely B Greenberg AJ Griffiths-Jones S Gross S Guigo R Gustafson EA Haerty W Hahn MW Halligan DL Halpern AL Halter GM Han MV Heger A Hillier L Hinrichs AS Holmes I Hoskins RA Hubisz MJ Hultmark D Huntley MA Jaffe DB Jagadeeshan S Jeck WR Johnson J Jones CD Jordan WC Karpen GH Kataoka E Keightley PD Kheradpour P Kirkness EF Koerich LB Kristiansen K Kudrna D Kulathinal RJ Kumar S Kwok R Lander E Langley CH Lapoint R Lazzaro BP Lee SJ Levesque L Li R Lin CF Lin MF Lindblad-Toh K Llopart A Long M Low L Lozovsky E Lu J Luo M Machado CA Makalowski W Marzo M Matsuda M Matzkin L McAllister B McBride CS McKernan B McKernan K Mendez-Lago M Minx P Mollenhauer MU Montooth K Mount SM Mu X Myers E Negre B Newfeld S Nielsen R Noor MA O'Grady P Pachter L Papaceit M Parisi MJ Parisi M Parts L Pedersen JS Pesole G Phillippy AM Ponting CP Pop M Porcelli D Powell JR Prohaska S Pruitt K Puig M Quesneville H Ram KR Rand D Rasmussen MD Reed LK Reenan R Reily A Remington KA Rieger TT Ritchie MG Robin C Rogers YH Rohde C Rozas J Rubenfield MJ Ruiz A Russo S Salzberg SL Sanchez-Gracia A Saranga DJ Sato H Schaeffer SW Schatz MC Schlenke T Schwartz R Segarra C Singh RS Sirot L Sirota M Sisneros NB Smith CD Smith TF Spieth J Stage DE Stark A Stephan W Strausberg RL Strempel S Sturgill D Sutton G Sutton GG Tao W Teichmann S Tobari YN Tomimura Y Tsolas JM Valente VL Venter E Venter JC Vicario S Vieira FG Vilella AJ Villasante A Walenz B Wang J Wasserman M Watts T Wilson D Wilson RK Wing RA Wolfner MF Wong A Wong GK Wu CI Wu G Yamamoto D Yang HP Yang SP Yorke JA Yoshida K Zdobnov E Zhang P Zhang Y Zimin AV Baldwin J Abdouelleil A Abdulkadir J Abebe A Abera B Abreu J Acer SC Aftuck L Alexander A An P Anderson E Anderson S Arachi H Azer M Bachantsang P Barry A Bayul T Berlin A Bessette D Bloom T Blye J Boguslavskiy L Bonnet C Boukhgalter B Bourzgui I Brown A Cahill P Channer S Cheshatsang Y Chuda L Citroen M Collymore A Cooke P Costello M D'Aco K Daza R De Haan G DeGray S DeMaso C Dhargay N Dooley K Dooley E Doricent M Dorje P Dorjee K Dupes A Elong R Falk J Farina A Faro S Ferguson D Fisher S Foley CD Franke A Friedrich D Gadbois L Gearin G Gearin CR Giannoukos G Goode T Graham J Grandbois E Grewal S Gyaltsen K Hafez N Hagos B Hall J Henson C Hollinger A Honan T Huard MD Hughes L Hurhula B Husby ME Kamat A Kanga B Kashin S Khazanovich D Kisner P Lance K Lara M Lee W Lennon N Letendre F LeVine R Lipovsky A Liu X Liu J Liu S Lokyitsang T Lokyitsang Y Lubonja R Lui A MacDonald P Magnisalis V Maru K Matthews C McCusker W McDonough S Mehta T Meldrim J Meneus L Mihai O Mihalev A Mihova T Mittelman R Mlenga V Montmayeur A Mulrain L Navidi A Naylor J Negash T Nguyen T Nguyen N Nicol R Norbu C Norbu N Novod N O'Neill B Osman S Markiewicz E Oyono OL Patti C Phunkhang P Pierre F Priest M Raghuraman S Rege F Reyes R Rise C Rogov P Ross K Ryan E Settipalli S Shea T Sherpa N Shi L Shih D Sparrow T Spaulding J Stalker J Stange-Thomann N Stavropoulos S Stone C Strader C Tesfaye S Thomson T Thoulutsang Y Thoulutsang D Topham K Topping I Tsamla T Vassiliev H Vo A Wangchuk T Wangdi T Weiand M Wilkinson J Wilson A Yadav S Young G Yu Q Zembek L Zhong D Zimmer A Zwirko Z Jaffe DB Alvarez P Brockman W Butler J Chin C Gnerre S Grabherr M Kleber M Mauceli E MacCallum I 2007 11 8 Evolution of genes and genomes on the Drosophila phylogeny. Nature 450 7167 0028-0836 203 218 10.1038/nature06341 17994087 Erlenbach T Haynes L Fish O Beveridge J Giambrone SA Reed LK Dyer KA Scott Chialvo CH 2023 12 13 Investigating the phylogenetic history of toxin tolerance in mushroom-feeding Drosophila. Ecol Evol 13 12 2045-7758 e10736 e10736 10.1002/ece3.10736 38099137 Gilbert LI 2004 2 27 Halloween genes encode P450 enzymes that mediate steroid hormone biosynthesis in Drosophila melanogaster. Mol Cell Endocrinol 215 1-2 0303-7207 1 10 10.1016/j.mce.2003.11.003 15026169 Gilbert LI Warren JT 2005 A molecular genetic approach to the biosynthesis of the insect steroid molting hormone. Vitam Horm 73 0083-6729 31 57 10.1016/S0083-6729(05)73002-8 16399407 Gramates LS Agapite J Attrill H Calvi BR Crosby MA Dos Santos G Goodman JL Goutte-Gattat D Jenkins VK Kaufman T Larkin A Matthews BB Millburn G Strelets VB the FlyBase Consortium. 2022 4 4 Fly Base: a guided tour of highlighted features. Genetics 220 4 0016-6731 10.1093/genetics/iyac035 35266522 Heed, W.B., Krishnamurthy, N.B. (1959). Genetic studies on the cardini group of Drosophila in the West Indies. University of Texas Publication 5914, 155-179. Heed, W.B. (1962) Genetic characteristics of island populations. University of Texas Publication 6205, 173-206. Jenkins VK Larkin A Thurmond J FlyBase Consortium 2022 Using FlyBase: A Database of Drosophila Genes and Genetics. Methods Mol Biol 2540 1064-3745 1 34 10.1007/978-1-0716-2541-5_1 35980571 Kent WJ Sugnet CW Furey TS Roskin KM Pringle TH Zahler AM Haussler D 2002 6 1 The human genome browser at UCSC. Genome Res 12 6 1088-9051 996 1006 10.1101/gr.229102 12045153 Kim BY Wang JR Miller DE Barmina O Delaney E Thompson A Comeault AA Peede D D'Agostino ERR Pelaez J Aguilar JM Haji D Matsunaga T Armstrong EE Zych M Ogawa Y Stamenković-Radak M Jelić M Veselinović MS Tanasković M Erić P Gao JJ Katoh TK Toda MJ Watabe H Watada M Davis JS Moyle LC Manoli G Bertolini E Košťál V Hawley RS Takahashi A Jones CD Price DK Whiteman N Kopp A Matute DR Petrov DA 2021 7 19 Highly contiguous assemblies of 101 drosophilid genomes. Elife 10 10.7554/eLife.66405 34279216 Larkin A Marygold SJ Antonazzo G Attrill H Dos Santos G Garapati PV Goodman JL Gramates LS Millburn G Strelets VB Tabone CJ Thurmond J FlyBase Consortium. 2021 1 8 FlyBase: updates to the Drosophila melanogaster knowledge base. Nucleic Acids Res 49 D1 0305-1048 D899 D907 10.1093/nar/gkaa1026 33219682 Li X Schuler MA Berenbaum MR 2007 Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics. Annu Rev Entomol 52 0066-4170 231 253 10.1146/annurev.ento.51.110104.151104 16925478 Lindberg RL Negishi M 1989 6 22 Alteration of mouse cytochrome P450coh substrate specificity by mutation of a single amino-acid residue. Nature 339 6226 0028-0836 632 634 10.1038/339632a0 2733794 Markow T. A. O’Grady P. 2008 9 20 Reproductive ecology of Drosophila Functional Ecology 22 5 0269-8463 747 759 10.1111/j.1365-2435.2008.01457.x Mudge JM Harrow J 2016 10 24 The state of play in higher eukaryote gene annotation. Nat Rev Genet 17 12 1471-0056 758 772 10.1038/nrg.2016.119 27773922 Obbard DJ Maclennan J Kim KW Rambaut A O'Grady PM Jiggins FM 2012 6 7 Estimating divergence dates and substitution rates in the Drosophila phylogeny. Mol Biol Evol 29 11 0737-4038 3459 3473 10.1093/molbev/mss150 22683811 Powell JR. (1997) Progress and prospects in evolutionary biology: the Drosophila model . New York: Oxford University Press. Rane RV Walsh TK Pearce SL Jermiin LS Gordon KH Richards S Oakeshott JG 2015 12 29 Are feeding preferences and insecticide resistance associated with the size of detoxifying enzyme families in insect herbivores? Curr Opin Insect Sci 13 70 76 10.1016/j.cois.2015.12.001 27436555 Raney BJ Barber GP Benet-Pagès A Casper J Clawson H Cline MS Diekhans M Fischer C Navarro Gonzalez J Hickey G Hinrichs AS Kuhn RM Lee BT Lee CM Le Mercier P Miga KH Nassar LR Nejad P Paten B Perez G Schmelter D Speir ML Wick BD Zweig AS Haussler D Kent WJ Haeussler M 2024 1 5 The UCSC Genome Browser database: 2024 update. Nucleic Acids Res 52 D1 0305-1048 D1082 D1088 10.1093/nar/gkad987 37953330 Raney BJ Dreszer TR Barber GP Clawson H Fujita PA Wang T Nguyen N Paten B Zweig AS Karolchik D Kent WJ 2013 11 13 Track data hubs enable visualization of user-defined genome-wide annotations on the UCSC Genome Browser. Bioinformatics 30 7 1367-4803 1003 1005 10.1093/bioinformatics/btt637 24227676 Ranson H Claudianos C Ortelli F Abgrall C Hemingway J Sharakhova MV Unger MF Collins FH Feyereisen R 2002 10 4 Evolution of supergene families associated with insecticide resistance. Science 298 5591 0036-8075 179 181 10.1126/science.1076781 12364796 Reis M Vieira CP Lata R Posnien N Vieira J 2018 12 1 Origin and Consequences of Chromosomal Inversions in the virilis Group of Drosophila. Genome Biol Evol 10 12 3152 3166 10.1093/gbe/evy239 30376068 Rele CP Sandlin KM Leung W Reed LK 2023 10 13 Manual annotation of Drosophila genes: a Genomics Education Partnership protocol. F1000Res 11 1579 1579 10.12688/f1000research.126839.3 37854289 Rendic S Di Carlo FJ 1997 5 1 Human cytochrome P450 enzymes: a status report summarizing their reactions, substrates, inducers, and inhibitors. Drug Metab Rev 29 1-2 0360-2532 413 580 10.3109/03602539709037591 9187528 Rewitz KF Rybczynski R Warren JT Gilbert LI 2006 12 1 The Halloween genes code for cytochrome P450 enzymes mediating synthesis of the insect moulting hormone. Biochem Soc Trans 34 Pt 6 0300-5127 1256 1260 10.1042/BST0341256 17073797 Robinson GE Hackett KJ Purcell-Miramontes M Brown SJ Evans JD Goldsmith MR Lawson D Okamuro J Robertson HM Schneider DJ 2011 3 18 Creating a buzz about insect genomes. Science 331 6023 0036-8075 1386 1386 10.1126/science.331.6023.1386 21415334 Russo CA Takezaki N Nei M 1995 5 1 Molecular phylogeny and divergence times of drosophilid species. Mol Biol Evol 12 3 0737-4038 391 404 10.1093/oxfordjournals.molbev.a040214 7739381 Scott JG 1999 9 1 Cytochromes P450 and insecticide resistance. Insect Biochem Mol Biol 29 9 0965-1748 757 777 10.1016/s0965-1748(99)00038-7 10510498 Stegeman JJ Livingstone DR 1998 11 1 Forms and functions of cytochrome P450. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 121 1-3 1367-8280 1 3 9972446 Stump AD Jablonski SE Bouton L Wilder JA 2011 12 1 Distribution and mechanism of α-amanitin tolerance in mycophagous Drosophila (Diptera: Drosophilidae). Environ Entomol 40 6 0046-225X 1604 1612 10.1603/EN11136 22217779 Sturtevant AH. (1916) Notes on North American Drosophilidae with descriptions of twenty-three new species. Annals of the Entomological Society of America 9(4): 323-343. Tamura K Subramanian S Kumar S 2003 8 29 Temporal patterns of fruit fly (Drosophila) evolution revealed by mutation clocks. Mol Biol Evol 21 1 0737-4038 36 44 10.1093/molbev/msg236 12949132 Tello-Ruiz MK Marco CF Hsu FM Khangura RS Qiao P Sapkota S Stitzer MC Wasikowski R Wu H Zhan J Chougule K Barone LC Ghiban C Muna D Olson AC Wang L Ware D Micklos DA 2019 10 28 Double triage to identify poorly annotated genes in maize: The missing link in community curation. PLoS One 14 10 e0224086 e0224086 10.1371/journal.pone.0224086 31658277 Threlfall J Blaxter M 2021 5 21 Launching the Tree of Life Gateway. Wellcome Open Res 6 2398-502X 125 125 10.12688/wellcomeopenres.16913.1 34095514 Warren JT Petryk A Marques G Jarcho M Parvy JP Dauphin-Villemant C O'Connor MB Gilbert LI 2002 8 12 Molecular and biochemical characterization of two P450 enzymes in the ecdysteroidogenic pathway of Drosophila melanogaster. Proc Natl Acad Sci U S A 99 17 0027-8424 11043 11048 10.1073/pnas.162375799 12177427 Williams E Chialvo P Scott Chialvo C 2026 5 27 Gene model for the ortholog of GstO3 in Drosophila dunni. MicroPubl Biol 2026 10.17912/micropub.biology.002110 42294398